Integrin heterodimer and a subunit thereof

ABSTRACT

A recombinant or isolated integrin heterodimer comprising a novel subunit α10 in association with a subunit β is described. The α10 integrin may be purified from bovine chondrocytes on a collagen-type-II affinity column. The integrin or the subunit α10 can be used as marker or target of all types of cells, e.g. of chondrocytes, osteoblasts and fibroblasts. The integrin or subunit α10 thereof can be used as marker or target in different physiological or therapeutic methods. They can also be used as active ingredients in pharmaceutical compositions and vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §§ 119 and/or 365 toInternational Application No. PCT/SE99/00544, filed on Mar. 31, 1999 andSwedish Patent Application Nos. 9801164-6 and 9900319-6 filed Apr. 2,1998 and Jan. 28, 1999, respectively.

FIELD OF THE INVENTION

The present invention relates to a recombinant or isolated integrinheterodimer comprising a subunit α10 and a subunit β, the subunit α10thereof, homologues and fragments of said integrin and of said subunitα10 having similar biological activity, processes of producing the same,polynucleotides and oligonucleotides encoding the same, vectors andcells comprising the same, binding entities binding specifically to thesame, and the use of the same.

BACKGROUND OF THE INVENTION

The integrins are a large family of transmembrane glycoproteins thatmediate cell-cell and cell-matrix interactions (1–5). All known membersof this superfamily are non-covalently associated heterodimers composedof an α- and β-subunit. At present, 8 β-subunits (β1–β8) (6) and 16α-subunits (α1–α9, αv, αM, αL, αX, αIIb, αE and αD) have beencharacterized (6–21), and these subunits associate to generate more than20 different integrins. The β1-subunit has been shown to associate withten different α-subunits, α1–α9 and αv, and to mediate interactions withextracellular matrix proteins such as collagens, laminins andfibronectin. The major collagen binding integrins are α1β1 and α2β1(22–25). The integrins α3β1 and α9β1 have also been reported to interactwith collagen (26, 27) although this interaction is not well understood(28). The extracellular N-terminal regions of the α and β integrinsubunits are important in the binding of ligands (29, 30). TheN-terminal region of the α-subunits is composed of a seven-fold repeatedsequence (12, 31) containing FG and GAP consensus sequences. The repeatsare predicted to fold into a β-propeller domain (32) with the last threeor four repeats containing putative divalent cation binding sites. Theα-integrin subunits α1, α2, αD, αE, αL, αM and αX contain a ˜200 aminoacid inserted domain, the I-domain (A-domain), which shows similarity tosequences in von Willebrand factor, cartilage matrix protein andcomplement factors C2 and B (33, 34). The I-domain is localized betweenthe second and third FG-GAP repeats, it contains a metal ion-dependentadhesion site (MIDAS) and it is involved in binding of ligands (35–38).

Chondrocytes, the only type of cells in cartilage, express a number ofdifferent integrins including α1β1, α2 β1, α3 β1, α5 β1, α6 β1, αvβ3,and αvβ5 (39–41). It has been shown that α1β1 and α2β1 mediatechondrocyte interactions with collagen type II (25) which is one of themajor components in cartilage. It has also been shown that α2β1 is areceptor for the cartilage matrix protein chondroadherin (42).

SUMMARY OF THE INVENTION

The present invention relates to a novel collagen type II bindingintegrin, comprising a subunit α10 in association with a subunit β,especially subunit β1, but also other β-subunits may be contemplated. Inpreferred embodiments, this integrin has been isolated from human orbovine articular chondrocytes, and human chondrosarcoma cells.

The invention also encompasses integrin homologues of said integrin,isolated from other species, such as bovine integrin heterodimercomprising a subunit α10 in association with a subunit β, preferably β1,as well as homologues isolated from other types of human cells or fromcells originating from other species.

The present invention relates in particular to a recombinant or isolatedintegrin subunit α10 comprising the amino acid sequence shown in SEQ IDNo. 1 or SEQ ID No. 2, and homologues and or fragments thereof havingthe same biological activity.

The invention further relates to a process of producing a recombinantintegrin subunit α10 comprising the amino acid sequence shown in SEQ IDNo. 1 or SEQ ID No. 2, or homologues or fragments thereof having similarbiological activity, which process comprises the steps of

-   -   a) isolating a polynucleotide comprising a nucleotide sequence        coding for a integrin subunit α10, or homologues or fragments        thereof having similar biological activity,    -   b) constructing an expression vector comprising the isolated        polynucleotide,    -   c) transforming a host cell with said expression vector,    -   d) culturing said transformed host cell in a culture medium        under conditions suitable for expression of integrin subunit        α10, or homologues or fragments thereof having similar        biological activity, in said transformed host cell, and,        optionally,    -   e) isolating the integrin subunit α10, or homologues or        fragments thereof having the same biological activity, from said        transformed host cell or said culture medium.

The integrin subunit α10, or homologues or fragments thereof having thesame biological activity, can also be provided by isolation from a cellin which they are naturally present.

The invention also relates to an isolated polynucleotide comprising anucleotide coding for a integrin subunit α10, or homologues or fragmentsthereof having similar biological activity, which polynucleotidecomprises the nucleotide sequence shown in SEQ ID No. 1 or SEQ ID No. 2,or parts thereof.

The invention further relates to an isolated polynucleotide oroligonucleotide which hybridises to a DNA or RNA encoding an integrinsubunit α10, having the amino acid sequence shown in SEQ ID No. 1 or SEQID No. 2, or homologues or fragments thereof, wherein saidpolyoligo-nucleotide or oligonucleotide fails to hybridise to a DNA orRNA encoding the integrin subunit α1.

The invention relates in a further aspect to vectors comprising theabove polynucleotides, and to cells containing said vectors and cellsthat have polynucleotides or oligonycleotides as shown in SEQ ID No. 1or 2 integrated in their genome.

The invention also relates to binding entities having the capability ofbinding specifically to the integrin subunit α10 or to homologues orfragments thereof, such as proteins, peptides, carbohydrates, lipids,natural ligands, polyclonal antibodies or monoclonal antibodies.

In a further aspect, the invention relates to a recombinant or isolatedintegrin heterodimer comprising a subunit α10 and a subunit β, in whichthe subunit α10 comprises the amino acid sequence shown in SEQ ID No. 1or SEQ ID No. 2, or homologues or fragments thereof having similarbiological activity.

In a preferred embodiment thereof, the subunit β is β1.

The invention also relates to a process of producing a recombinantintegrin heterodimer comprising a subunit α10 and a subunit β, in whichthe subunit α10 comprises the amino acid sequence shown in SEQ ID No. 1or SEQ ID No. 2, which process comprises the steps of

-   -   a) isolating one polynucleotide comprising a nucleotide sequence        coding for a subunit α10 of an integrin heterodimer and,        optionally, another polynucleotide comprising a nucleotide        sequence coding for a subunit β of an integrin heterodimer, or        for homologues or fragments thereof having similar biological        activity,    -   b) constructing an expression vector comprising said isolated        polynucleotide coding for said subunit α10 in combination with        an expression vector comprising said isolated nucleotide coding        for said subunit β,    -   c) transforming a host cell with said expression vectors,    -   d) culturing said transformed host cell in a culture medium        under conditions suitable for expression of an integrin        heterodimer comprising a subunit α10 and a subunit β, or        homologues or fragments thereof similar biological activity, in        said transformed host cell, and, optionally,    -   e) isolating the integrin heterodimer comprising a subunit α10        and a subunit β, or homologues or fragments thereof having the        same biological activity, from said transformed host cell or        said culture medium.

The integrin heterodimer, or homologues or fragments thereof havingsimilar biological activity, can also be provided by isolation from acell in which they are naturally present.

The invention further relates to a cell containing a first vector, saidfirst vector comprising a polynucleotide coding for a subunit α10 of anintegrin heterodimer, or for homologues or parts thereof having similarbiological activity, which polynucleotide comprises the nucleotidesequence shown in SEQ ID No. 1 or SEQ ID No. 2 or parts thereof, and,optionally, a second vector, said second vector comprising apolynucleotide coding for a subunit β of an integrin heterodimer, or forhomologues or fragments thereof.

In still another aspect, the invention relates to binding entitieshaving the capability of binding specifically to the integrinheterodimer comprising a subunit α10 and a subunit β, or to homologuesor fragments thereof having similar biological activity, preferablywherein the subunit β is β1. Preferred binding entities are proteins,peptides, carbohydrates, lipids, natural ligands, polyclonal antibodiesand monoclonal antibodies.

In a further aspect, the invention relates to a fragment of the integrinsubunit α10, which fragment is a peptide chosen from the groupcomprising peptides of the cytoplasmic domain, the I-domain and thespliced domain.

In one embodiment, said fragment is a peptide comprising the amino acidsequence KLGFFAHKKIPEEEKREEKLEQ (SEQ ID NO: 7).

In another embodiment, said fragment comprises the amino acid sequencefrom about amino acid no. 952 to about amino acid no. 986 of SEQ ID No.1.

In a further embodiment, said fragment comprises the amino acid sequencefrom about amino acid No. 140 to about amino acid No. 337 in SEQ ID No.1.

Another embodiment of the invention relates to a polynucleotide oroligonucleotide coding for a fragment of the human integrin subunit α10.In one embodiment this polynucleotide of oligonucleotide codes for afragment which is a peptide chosen from the group comprising peptides ofthe cytoplasmic domain, the I-domain and the spliced domain. In furtherembodiments the polynucleotide or oligonucleotide codes for thefragments defined above.

The invention also relates to binding entities having the capability ofbinding specifically to a fragment of the integrin subunit α10 asdefined above.

The invention also relates to a process of using an integrin subunit α10comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.2, or an integrin heterodimer comprising said subunit α10 and a subunitβ, or a homologue or fragment of said integrin or subunit having similarbiological activity, as a marker or target molecule of cells or tissuesexpressing said integrin subunit α10, which cells or tissues are ofanimal including human origin.

In an embodiment of this process the fragment is a peptide chosen fromthe group comprising peptides of the cytoplasmic domain, the I-domainand the spliced domain.

In further embodiments of said process the fragment is a peptidecomprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ (SEQ ID NO:7), or a fragment comprising the amino acid sequence from about aminoacid No. 952 to about amino acid No. 986 of SEQ ID No. 1, or a fragmentcomprising the amino acid sequence from about amino acid no. 140 toabout amino acid no. 337 of SEQ ID no. 1.

The subunit β is preferably β1. The cells are preferably chosen from thegroup comprising chondrocytes, smooth muscle cells, endothelial cells,osteoblasts and fibroblasts.

Said process may be used during pathological conditions involving saidsubunit α10, such as pathological conditions comprising damage ofcartilage, or comprising trauma, rheumatoid arthritis andosteoarthritis.

Said process may be used for detecting the formation of cartilage duringembryonal development, or for detecting physiological or therapeuticreparation of cartilage.

Said process may also be used for selection and analysis, or forsorting, isolating or purification of chondrocytes.

A further embodiment of said process is a process for detectingregeneration of cartilage or chondrocytes during transplantation ofcartilage or chondrocytes.

A still further embodiment of said process is a process for in vitrostudies of differentiation of chondrocytes.

The invention also comprises a process of using binding entities havingthe capability of binding specifically to an integrin subunit α10comprising the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No.2, or an integrin heterodimer comprising said subunit α10 and a subunitβ, or to homologues or fragments thereof having similar biologicalactivity, as markers or target molecules of cells or tissues expressingsaid integrin subunit α10, which cells or tissues are of animalincluding human origin.

The fragment in said process may be a peptide chosen from the groupcomprising peptides of the cytoplasmic domain, the I-domain and thespliced domain. In preferred embodiments said fragment is a peptidecomprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ (SEQ ID NO:7), or a fragment comprising the amino acid sequence from about aminoacid No. 952 to about amino acid No. 986 of SEQ ID No. 1, or a fragmentcomprising the amino acid sequence from about amino acid No. 140 toabout amino acid no. 337 of SEQ ID No. 1.

The process may also be used for detecting the presence of an integrinsubunit α10 comprising the amino acid sequence shown in SEQ ID No. 2, orof an integrin heterodimer comprising said subunit α10 and a subunit β,or of homologues or fragments thereof having similar biologicalactivity.

In a further embodiment said process is a process for determining thedifferentiation-state of cells during embryonic development,angiogenesis, or development of cancer.

In a still further embodiment this process is a process for detectingthe presence of an integrin subunit α10, or a homologue or fragment ofsaid integrin subunit having similar biological activity, on cells,whereby a polynucleotide or oligonucleotide chosen from the groupcomprising a polynucleotide or oligonucleotide chosen form thenucleotide sequence shown in SEQ ID No. 1 is used as a marker underhybridization conditions wherein said polynucleotide or oligonucleotidefails to hybridise to a DNA or RNA encoding an integrin subunit α1. Saidcells may be chosen from the group comprising chondrocytes, smoothmuscle cells, endothelial cells, osteoblasts and fibroblasts. Saidintegrin fragment may be a peptide chosen form the group comprisingpeptides of the cytoplasmic domain, the I-domain and the spliced domain,such as a peptide comprising the amino acid sequenceKLGFFAHKKIPEEEKREEKLEQ (SEQ ID NO: 7), or a fragment comprising theamino acid from about amino acid no. 952 to about amino acid no. 986 ofSEQ ID No. 1, or a fragment comprising the amino acid sequence fromabout amino acid No. 140 to about amino acid no. 337 of SEQ ID No. 1.

In a still further embodiment the process is a process for determiningthe differentiation-state of cells during development, in pathologicalconditions, in tissue regeneration or in therapeutic and physiologicalreparation of cartilage. The pathological conditions may be anypathological conditions involving the integrin subunit α10, such asrheumatoid arthritis, osteoarthrosis or cancer. The cells may be chosenfrom the group comprising chondrocytes, smooth muscle cells, endothelialcells, osteoblasts and fibroblasts.

The invention also relates to a process for determining thedifferentiation-state of cells during development, in pathologicalconditions, in tissue regeneration and in therapeutic and physiologicalreparation of cartilage, whereby a polynucleotide or oligonucleotide isused as a marker under hybridization conditions wherein saidpolynucleotide or oligonucleotide is a polynucleotide or ologonucleotidecoding for a peptide plasmic domain, the I-domain and the spliceddomain, such as a polynucleotide or oligonucleotide coding for a peptidecomprising the amino acid sequence KLGFFAHKKIPEEEKREEKLEQ (SEQ ID NO:7), or comprising the amino acid sequence from about amino acid No. 952to about amino acid no. 986 of SEQ ID No. 1, or the amino acid sequenceform about amino acid No. 140 to about amino acid No. 337 of SEQ IDNo. 1. Said pathological conditions may be any pathological conditionsinvolving the integrin subunit cancer, or atheroclerosis orinflammation. Said cells may be chosen from the group comprisingchondrocytes, smooth nuscle cells, endothelial cells, osteoblasts andfibroblasts.

In a further aspect the invention relates to a pharmaceuticalcomposition comprising as an active ingredient a pharmaceutical agent oran antibody which is capable of using an integrin heterodimer comprisinga subunit α10 and a subunit β, or the subunit α10 thereof, or ahomologue or fragment of said integrin or subunit α10 having similarbiological activity, as a target molecule. An embodiment of saidpharmaceutical composition is intended for use in stimulating,inhibiting or blocking the formation of cartilage, bone or bloodvessels. A further embodiment comprises a pharmaceutical composition foruse in preventing adhesion between tendon/ligaments and the surroundingtissue after infection, inflammation and after surgical interventionwhere adhesion impairs the function of the tissue.

The invention is also related to a vaccine comprising as an activeingredient an integrin heterodimer comprising a subunit α10 and asubunit β, or the subunit α10 thereof, or a homologue or fragment ofsaid integrin or subunit α10, or DNA or RNA coding for said integrinsubunit α10.

A further aspect of the invention is related to the use of the integrinsubunit α10 as defined above as a marker or target in transplantation ofcartilage or chondrocytes.

A still further aspect of the invention is related to a method of usingbinding entities having the capability of binding specifically to anintegrin subunit α10 comprising the amino acid sequence shown in SEQ IDNo. 1 or SEQ ID No. 2, or an integrin heterodimer comprising saidsubunit α10 and a subunit β, or to homologues or fragments thereofhaving similar biological activity, for promoting adhesion ofchondrocytes and/or osteoblasts to surfaces of implants to stimulateosseointegration.

The invention is also related to the use of an integrin subunit α10 oran integrin heterodimer comprising said subunit α10 and a subunit βas atarget for anti-adhesive drugs or molecules in tendon, ligament,skeletal muscle or other tissues where adhesion impairs the function ofthe tissue.

The invention also relates to a method of stimulating, inhibiting orblocking the formation of cartilage or bone, comprising administrationto a subject a suitable amount of a pharmaceutical agent or an antibodywhich is capable of using an integrin heterodimer comprising a subunitα10 and a subunit β, or the subunit α10 thereof, or a homologue orfragment of said integrin or subunit α10 having similar biologicalactivity, as a target molecule.

In another embodiment the invention is related to a method of preventingadhesion between tendon/ligaments and the surrounding tissue afterinfection, inflammation and after surgical intervention where adhesionimpairs the function of the tissue, comprising administration to asubject a suitable amount of a pharmaceutical agent or an antibody whichis capable of using a integrin heterodimer comprising a subunit α10 anda subunit β, or the subunit α10 thereof, or a homologue or fragment ofsaid integrin or subunit α10 having similar biological activity, as atarget molecule.

The invention also relates to a method of stimulating extracellularmatrix synthesis and repair by activation or blockage of an integrinheterodimer comprising a subunit α10 and a subunit β, or of the subunitα10 thereof, or of a homologue or fragment of said integrin or subunitα10 having similar biological activity.

In a further aspect the invention relates to a method of in vitrodetecting the presence of integrin binding entities, comprisinginteraction of an integrin heterodimer comprising a subunit α10 and asubunit β, or the subunit α10 thereof, or a homologue or fragment ofsaid integrin or subunit, with a sample, thereby causing said integrin,subunit α10, or homologue or fragment thereof having similar biologicalactivity, to modulate the binding to its natural ligand or otherintegrin binding proteins present in said sample.

The invention also relates to a method of in vitro studying consequencesof the interaction of a human heterodimer integrin comprising a subunitα10 and a subunit β, or the subunit α10 thereof, or a homologue orfragment of said integrin or subunit, with an integrin binding entityand thereby initiate a cellular reaction. Said consequences may bemeasured as alterations in cellular functions.

A still further aspect of the inventions relates to a method of usingDNA or RNA encoding an integrin subunit α10 or homologues or fragmentsthereof as a molecular target. In an embodiment of this aspect, apolynucleotide or oligonucleotide hybridises to the DNA or RNA encodingan integrin subunit α10 or homologues or fragments thereof, whereby saidpolynucleotide or oligonucleotide fails to hybridise to a DNA or RNAencoding en integrin subunit α1.

The invention also relates to a method of using a human heterodimerintegrin comprising a subunit α10 and a subunit β, or the subunit α10thereof, or a homologue or fragment of said integrin or subunit, or aDNA or RNA encoding an integrin subunit α10 or homologues or fragmentsthereof, as a marker or target molecule during angiogenesis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Affinity purification of the α10 integrin subunit on collagentype II-Sepharose.

FIG. 2. Amino acid sequences of peptides from the bovine α10 integrinsubunit (SEQ ID NOS: 26–31, respectively, in order of appearance).

FIG. 3 a. Affinitypurification and immunoprecipitation of the integrinsubunit α10 from bovine chondrocytes.

FIG. 3 b. Affinitypurification and immunoprecipitation of the integrinsubunit α10 from human chondrocytes.

FIG. 3 c. Affinitypurification and immunoprecipitation of the integrinsubunit α10 from human chondrosarcoma cells.

FIG. 4. A 900 bp PCR-fragment corresponding to the bovine integrinsubunit α10.

FIG. 5. Schematic map of the three overlapping α10 clones.

FIG. 6. Nucleotide sequence (SEQ ID NO: 1) and deduced amino acidsequence (SEQ ID NO: 4) of the human α10 integrin subunit.

FIG. 7. Northern blot of integrin α10 mRNA.

FIG. 8 Immunoprecipitation of the α10 integrin subunit from humanchondrocytes using antibodies against the cytoplasmic domain of α10 (a).Western blot of the α10 associated β-chain (b).

FIG. 9. Immunostaining of α10 integrin in human articular cartilage.

FIG. 10 Immunostaining of α10 integrin in 3 day mouse limb cartilage.

FIG. 11. Immunostaining of α10 integrin in 13.5 day mouse embryo.

FIG. 12. Hybridisation of α10 mRNA in various human tissues.

FIG. 13 Immunostaining of fascia around tendon (a), skeletal muscle (b)and heart valves (c) in 3 day mouse limb.

FIG. 14. PCR fragments corresponding to α10 integrin subunit from humanchondrocytes, human endothelial cells, human fibroblasts and rat tendon.

FIG. 15. Partial genomic nucleotide sequence (SEQ ID NO: 32) of thehuman integrin subunit α10 (Top protein sequence disclosed as SEQ IDNOS: 33–127; middle protein sequence disclosed as SEQ ID NOS: 128–206;bottom protein sequence disclosed as SEQ ID NOS: 207–299).

FIG. 16. Upregulation of α10 integrin subunit in chondrocytes culturedin alginate.

FIG. 17. Immunoprecipitation of the α10 integrin subunit from humansmooth muscle cells

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrate that human and bovine chondrocytesexpress a novel, collagen type II-binding integrin in the β1-family. Anearlier study presented some evidence for that human chondrosarcomacells also express this integrin (25). Immunoprecipitation experimentsusing antibodies against the integrin subunit β1 revealed that thisnovel α-integrin subunit had an apparent molecular weight (M_(r)) ofapproximately 160 kDa under reducing conditions, and was slightly largerthan the α2 integrin subunit. To isolate this α-subunit collagen typeII-binding proteins were affinity purified from bovine chondrocytes. Thechondrocyte lysate was first applied to a fibronectin-Sepharoseprecolumn and the flow through was then applied to a collagen typeII-Sepharose column. A protein with M_(r) of approximately 160 kD wasspecifically eluted with EDTA from the collagen column but not from thefibronectin column. The M_(r) of this protein corresponded with theM_(r) of the unidentified β1-related integrin subunit. The 160 kDprotein band was excised from the SDS-PAGE gel, digested with trypsinand the amino acid sequences of the isolated peptides were analysed.

Primers corresponding to isolated peptides amplified a 900 bpPCR-fragment from bovine cDNA which was cloned, sequenced and used forscreening of a human articular chondrocyte λZapII cDNA library to obtainthe human integrin α-subunit homologue. Two overlapping clones, hc1 andhc2 were isolated, subcloned and sequenced. These clones contained ⅔ ofthe nucleotide sequence including the 3′ end of the cDNA. A third clonewhich contained the 5′ end of the α10 cDNA, was obtained using the RACEtechnique. Sequence analysis of the 160 kD protein sequence showed thatit was a member of the integrin α-subunit family and the protein wasnamed α10.

The deduced amino acid sequence of α10 was found to share the generalstructure of the integrin α-subunits described in previously publishedreports (6–21). The large extracellular N-terminal part of α10 containsa seven-fold repeated sequence which was recently predicted to fold intoa β-propeller domain (32). The integrin subunit α10 contains threeputative divalent cation-binding sites (DxD/NxD/NxxxD) (53), a singlespanning transmembrane domain and a short cytoplasmic domain. Incontrast to most α-integrin subunits the cytoplasmic domain of α10 doesnot contain the conserved sequence KxGff(R/K) R (SEQ ID NO: 22). Thepredicted amino acid sequence in α10 is KLGFFAH (SEQ ID NO: 8). Severalreports indicate that the integrin cytoplasmic domains are crucial insignal transduction (54) and that membrane-proximal regions of both α-and β-integrin cytoplasmic domains are involved in modulatingconformation and affinity state of integrins (55–57). It is suggestedthat the GFFKR (SEQ ID NO: 23) motif in α-chains are important forassociation of integrin subunits and for transport of the inegrin to theplasma membrane (58). The KxGFFKR (SEQ ID NO: 24) domain has been shownto interact with the intracellular protein calreticulin (59) andinterstingly, calreticulin-null embryonic stem cells are deficient inintegrin-mediated cell adhesion (60). It is therefore possible that thesequence KLGFFAH (SEQ ID NO: 8) in α10 have a key function in regulatingthe affinity between α10β1 and matrix proteins.

Integrin α subunits are known to share an overall identity of 20–40%(61). Sequence analysis showed that the α10 subunit is most closelyrelated to the I-domain containing α-subunits with the highest identityto α1 (37%) and α2 (35%). The integrins α1β1 and α2β1 are knownreceptors for both collagens and laminins (24; 62; 63) and we have alsorecently demonstrated that α2β1 interacts with the cartilage matrixprotein chondroadherin (42). Since α10β1 was isolated on a collagen typeII-Sepharose, we know that collagen type II is a ligand for α10β1. Wehave also shown by affinity purification experiments that α10β1interacts with collagen type I but it remains to be seen whether lamininor chondroadherin are also ligands for this integrin.

The α10 associated β-chain migrated as the β1 integrin subunit bothunder reducing and non-reducing conditions. To verify that the α10associated β-chain indeed is β1, chondrocyte lysates wereimmunoprecipitated with antibodies against α10 or β1 followed by Westernblot using antibodies against the β1-subunit. These results clearlydemonstrated that α10 is a member of the β1-integrin family. However,the possibility that α10 combine also with other β-chains can not beexcluded.

A polyclonal peptide antibody raised against the cytoplasmic domain ofα10 precipitated two protein bands with M_(r) of approximately 160 kD(α10) and 125 kD (β1) under reducing conditions. Immunohistochemistryusing the α10-antibody showed staining of the chondrocytes in tissuesections of human articular cartilage. The antibody staining was clearlyspecific since preincubation of the antibody with the α10-peptidecompletely abolished the staining. Immunohistochemical staining of mouselimb sections from embryonic tissue demonstrated that α10 is upregulatedduring condensation of the mesenchyme. This indicate that the integrinsubunit α10 is important during the formation of cartilage. In 3 day oldmice α10 was found to be the dominating collagen binding integrinsubunit which point to that α10 has a key function in maintaining normalcartilage functions.

Expression studies on the protein and mRNA level show that thedistribution of α10 is rather restrictive. Immunohistochemistry analyseshave shown that α10 integrin subunit is mainly expressed in cartilagebut it is also found in perichondrium, periosteum, ossification grooveof Ranvier, in fascia surrounding tendon and skeletal muscle and in thetendon-like structures in the heart valves. This distribution point tothat α10 integrin subunit is present also on fibroblasts andosteoblasts. PCR amplification of cDNA from different cell typesrevealed the presence of an alternatively spliced α10 integrin subunit.This spliced α10 was dominating in fibroblasts which suggests that α10in fibroblasts may have a different function compared to α10 present onchondrocytes.

Expression of the integrin subunit α10 was found to decrease whenchondrocytes were cultured in monolayer. In contrast, the expression ofα10 was found to increase when the cells were cultured in alginatebeads. Since the latter culturing model is known to preserve thephenotype of chondrocytes the results suggest that α10 can function asmarker for a differentiated chondrocyte.

Adhesion between tendon/ligaments and the surrounding tissue is awell-known problem after infection, injury and after surgicalintervention. Adhesion between tendon and tendon sheets impairs thegliding function and cause considerable problems especially duringhealing of tendons in e.g. the hand and fingers leading to functionalincapacity. The localisation of the α10 integrin subunit in the fasciaof tendon and skeletal muscle makes α10 a possible target for drugs andmolecules with anti-adhesive properties that could prevent impairment ofthe function of tendon/ligament. The integrin subunit α10 can also be atarget for drugs or molecules with anti-adhesive properties in othertissues where adhesion is a problem.

EXAMPLES Example 1

Affinity purification of the α10integrin subunit on collagen typeII-Sepharose.

Materials and Methods

Bovine chondrocytes, human chondrocytes or human chondrosarcoma cellswere isolated as described earlier [Holmvall et al, Exp Cell Res, 221,496–503 (1995), Camper et al, JBC, 273, 20383–20389 (1998)]. A TritonX-100 lysate of bovine chondrocytes was applied to afibronectin-Sepharose precolumn followed by a collagen type II-Sepharosecolumn and the integrin subunit α10 was eluted from the collagen typeII-column by EDTA (Camper et al, JBC, 273, 20383–20389 (1998). Theeluted proteins were precipitated by methanol/chloroform, separated bySDS-PAGE under reducing conditions and stained with Coomassie blue.(Camper et al, JBCu, 273, 20383–20389 (1998). Peptides from the α10protein band were isolated by in-gel digestion with a trypsin and phaseliquid chromatography and sequenced by Edman degradation (Camper et al,JBC, 273, 20383–20389 (1998).

Results

FIG. 1 shows EDTA-eluted proteins from the fibronectin-Sepharose (A),flow-through from the collagen type II-Sepharose column (B) andEDTA-eluted proteins from the collagen type II-Sepharose (C). The α10integrin subunit (160 kDa) which was specifically eluted from thecollagen type II column is indicated with an arrow. FIG. 2 shows theamino acid sequences of six peptides that were isolated from the bovineintegrin subunit α10. FIGS. 3 a, b, and c show that the α10 integrinsubunit is present on bovine chondrocytes (3 a), human chondrocytes (3b) and human chondrosarcoma cells (3 c). The affinity for collagen typeII, the coprecipitation with β1-integrin subunit and the molecularweight of 160 kDa under reducing conditions identify the α10 integrinsubunit on the different cells. These results show that α10 can beisolated from chondrocytes and from chondrosarcoma cells.

Example 2

Amplification of PCR fragment corresponding to bovine α10 integrinsubunit.

Materials and Methods

The degenerate primers GAY AAY ACI GCI CAR AC (SEQ ID NO: 9) (DNTAQT,SEQ ID NO: 10, forward) and TIA TIS WRT GRT GIG GYT (SEQ ID NO: 11)(EPHHSl, SEQ ID NO: 12, reverse) were used in PCR (Camper et al, JBC,273, 20383–20389 (1998) to amplify the nucleotide sequence correspondingto the bovine peptide 1 (FIG. 2). A 900 bp PCR-fragment was thenamplicfied from bovine cDNA using an internal specific primer TCA GCCTAC ATT CAT TAT (SEQ ID NO: 13) (SAYIQY, SEQ ID NO: 14, forward)corresponding to the cloned nucleotide sequence of peptide 1 togetherwith the generate primer ICK RTC CCA RTG ICC IGG (SEQ ID NO: 15)(PGHWDP, SEQ ID NO: 16, reverse) corresponding to the bovine peptide 2(FIG. 2). Mixted bases were used in positions that were twofolddegenerate and inosines were used in positions that are three- orfourfold degenerate. mRNA isolation and cDNA synthesis was done asearlier described (Camper et al, JBC, 273, 20383–20389 (1998)).

Results

The nucleotide sequence of peptide 1 (FIG. 2) was obtained byPCR-amplification, cloning and sequencing of bovine cDNA. From thisnucleotide sequence an exact primer was designed and applied inPCR-amplification with degenerate primers corresponding to peptides 2–6(FIG. 2). Primers corresponding to peptides 1 and 2 amplified a 900 bpPCR-fragment from bovine cDNA (FIG. 4).

Example 3

Cloning and sequence analysis of the human α10 integrin subunit.

Material and Methods

The cloned 900 bp PCR-fragment, corresponding to bovine α10-integrin,was digoxigenin-labelled according to the DIG DNA labeling kit(Boehringer Mannheim) and used as a probe for screening of a humanarticular chondrocyte λZapII cDNA library (provided by Michael Bayliss,The Royal Veterinary Basic Sciences, London, UK)(52). Positive clonescontaining the pBluescript SK+ plasmid with the cDNA insert were rescuedfrom the ZAP vector by in vivo excision as described in the ZAP-cDNA®synthesis kit (Stratagene). Selected plasmids were purified andsequenced as described earlier (Camper et al, JBC, 273, 20383–20389(1998)) using T3, T7 and internal specific primers. To obtain cDNA thatencoded the 5′ end of al 0 we designed the primer AAC TCG TCT TCC AGTGCC ATT CGT GGG (SEQ ID NO: 17, reverse; residue 1254–1280 in α10 cDNA)and used it for rapid amplification of the cDNA 5′ end (RACE) asdescribed in the Marathon™ cDNA Amplification kit (Clontech INC., PaloAlto, Calif.).

Results

Two overlapping clones, hc1 and hc2 (FIG. 5), were isolated, subclonedand sequenced. These clones contained ⅔ of the nucleotide sequenceincluding the 3′ end of the cDNA. A third clone (race1; FIG. 5), whichcontained the 5′ end of the α10 cDNA, was obtained using the RACEtechnique. From these three overlapping clones of α10 cDNA, 3884nucleotides were sequenced. The nucleotide sequence and deduced aminoacid sequence is shown in FIG. 6. The sequence contains a3504-nucleotide open reading frame that is predicted to encode a 1167amino acid mature protein. The signal peptide cleavage site is markedwith an arrow, human homologues to bovine peptide sequences areunderlined and the I-domain is boxed. Metal ion binding sites areindicated with a broken underline, potential N-glycosylation sites areindicated by an asterisk and the putative transmembrane domain is doubleunderlined. The normally conserved cytoplasmic sequence is indicated bya dot and dashed broken underline.

Sequence analysis demonstrate that α10 is a member of the integrinα-subunit family.

Example 4

Identification of a clone containing a splice variant of α10.

One clone which was isolated from the human chondrocyte library (seeExample 3) contained a sequence that was identical to the sequence ofα10 integrin subunit except that the nucleotides between nt positions2942 and 3055 were deleted. The splice variant of α10 was verified inPCR experiment using primers flanking the splice region (see FIG. 14).

Example 5

Identification of α10 integrin subunit by Northern blot.

Material and Methods

Bovine chondrocyte mRNA was purified using a QuickPrep®Micro mRNAPurification Kit (Pharmacia Biotech, Uppsala, Sweden), separated on a 1%agarose-formaldehyde gel, transferred to nylon membranes and immobilisedby UV crosslinking. cDNA-probes were 32P-labelled with Random Primed DNALabeling Kit (Boehringer Mannheim). Filters were prehybridised for 2–4hours at 42° C. in 5×SSE, 5× Denharts solution, 0.1% SDS, 50 μg/mlsalmon sperm DNA and 50% formamide and then hybridised over night at 42°C. with the same solution containing the specific probe (0.5–1×106cpm/ml). Specifically bound cDNA-probes were analysed using thephosphoimager system (Fuji). Filters were stripped by washing in 0.1%SDS, for 1 hour at 80° C. prior to re-probing. The α10-integrincDNA-probe was isolated from the race1-containing plasmid using therestriction enzymes BamHI (GIBCO BRL) and NcoI (Boehringer Mannheim).The rat β1-integrin cDNA probe was a kind gift from Staffan Johansson,Uppsala, Sweden.

Results

Northern blot analysis of mRNA from bovine chondrocytes showed that ahuman α10 cDNA-probe hybridised with a single mRNA of approximately 5.4kb (FIG. 7). As a comparison, a cDNA-probe corresponding to the integrinsubunit α1 was used. This cDNA-probe hybridised a mRNA-band ofapproximately 3.5 kb on the same filter. These results show that acDNA-probe against α10 can be used to identify the α10 integrin subuniton the mRNA level.

Example 6

Preparation of antibodies against the integrin subunit α10.

A peptide corresponding to part of the α10 cytoplasmic domain,Ckkipeeekreekle (SEQ ID NO: 25, see FIG. 6) was synthesized andconjugated to keyhole limpet hemocyanin (KLH). Rabbits were immunizedwith the peptide-KLH conjugate to generate antiserum against theintegrin subunit α10. Antibodies recognizing α10 were affinity purifiedon an peptide-coupled column (Innovagen AB).

Example 7

Immunoprecipitation of the integrin subunit α10 from chondrocytes.

Material and Methods

Human chondrocytes were ¹²⁵I-labelled, lyzed with Triton X-100 andimmunoprecipitated as earlier described (Holmvall et al, Exp Cell Res,221, 496–503 (1995), Camper et al, JBC, 273, 20383–20389 (1998)). TritonX-100 lysates of 125I-labeled human chondrocytes were immunoprecipitatedwith polyclonal antibodies against the integrin subunits β1, α1, α2, α3or α10. The immunoprecipitated proteins were separated by SDS-PAGE(4–12%) under non-reducing conditions and visualised using aphospho-imager. Triton X-100 lysates of human chondrocytesimmunoprecipitated with α10 or β1 were separated by SDS-PAGE (8%) undernon-reducing conditions and analysed by Western blot using thepolyclonal β1 antibody and chemiluminescent detection as described inCamper et al, JBC, 273, 20383–20389 (1998).

Results

The polyclonal peptide antibody, raised against the cytoplasmic domainof α10, precipitated two protein bands with Mr of approximately 160 kD(α10) and 125 kD (β1) under reducing conditions. The α10 associatedβ-chain migrated as the β1 integrin subunit (FIG. 8 a). To verify thatthe α10 associated β-chain in chondrocytes indeed is β1, chondrocytelysates were immunoprecipitated with antibodies against α10 orb β1followed by Western blot using antibodies against the β1-subunit (FIG. 8b). These results clearly demonstrated that α10 is a member of theβ1-integrin family. However, the results do not exclude the possibilitythat α10 can associate with other β-chains in other situations.

Example 8

Immunohistochemical staining of the integrin subunit α10 in human andmouse cartilage.

Material and Methods

Frozen sections of adult cartilage (trochlear groove) obtained duringsurgery (provided by Anders Lindahl, Salgrenska Hospital, Gothenburg,Sweden and frozen sections from of 3 day old mouse limb were fixed andprepared for immunohistochemistry as earlier described (Camper et al,JBC, 273, 20383–20389 (1998)). Expression of α10 integrin subunit wasanalysed using the polyclonal antibody against the cytoplasmic domain asa primary antibody (see Example 6) and a secondary antibody conjugatedto peroxidase.

Results

FIG. 9 show immunostaining of human adult articular cartilage.

The α10-antibody recognising the cytoplasmic domain of α10 stained thechondrocytes in tissue sections of human articular: cartilage (A). Thestaining was depleted when the antibody was preincubated with theα10-peptide (B). A control antibody recognising the α9 integrin subunitdid not bind to the chondrocyte (C).

FIG. 10 shows that the α10 antibody stain the majority of chondrocytesin the growing bone anlage (a and b). The α10 antibody also recognisedcells in the ossification groove of Ranvier (b), especially theosteoblast in the bone bark which are lining the cartilage in themetaphys are highly positive for α10. The cells in the ossificationgroove of Ranvier are believed to be important for the growth indiameter of the bone. The integrin subunit α10 is also highly expressedin perichondrium and periosteum. Cell in these tissues are likelyimportant in the repair of the cartilage tissue. The describedlocalisation of the integrin subunit α10 suggest that this integrin isimportant for the function of the cartilage tissue.

Example 9

Immunohistochemical staining of the integrin subunit α10 during mousedevelopment.

Material and Methods

Frozen sections from mouse embryos (13.5 days) were investigated forexpression of α10 by immunhistochemistry as described in Camper et al,JBC, 273, 20383–20389 (1998). Expression of α10 integrin subunit wasanalysed using the polyclonal antibody against the cytoplasmic domain asa primary antibody (see Example 6) and a secondary antibody conjugatedto peroxidase. The embryo sections were also investigated for expressionof integrin subunit α1 (monoclonal antibody from Pharmingen) andcollagen type II (monoclonal antibody, kind gift from Dr John Mo, LundUniversity, Sweden).

Results

FIG. 11 show that α10 integrin subunit is unregulated in the limb whenthe mesenchymal cells undergo condensation to form cartilage (a).Especially the edge of the newly formed cartilage has high expression ofα10. The formation of cartilage is verified by the high expression ofthe cartilage specific collage type II (b). The control antibody againstα1 integrin subunit showed only weak expression on the cartilage (c). Inother experiments expression of α10 was found in all cartilagecontaining tissues in the 3 day old mouse including limbs, ribs andvertebrae. The upregulation of α10 during formation of cartilage suggestthat this integrin subunit is important both in the development ofcartilage and bone and in the repair of damaged cartilage tissue.

Example 10

mRNA expression of α10 in tissues other than articular cartilage.

Material and Methods

Expression of α10 integrin subunit was examined on the mRNA level indifferent human tissues. A Northern dot blot with immobilised mRNA fromthe listed tissues in FIG. 12 was hybridised with an α10 integrin cDNAprobe isolated from the race 1-containing plasmid using the restrictionenzymes BamH1 and Nco1. The degree of hybridisation was analysed using aphospho imager. The following symbols denote mRNA level in increasingorder: −, +, ++, +++, ++++.

Results

Analysis of the hybridised mRNA showed that α10 was expressed in aorta,trachea, spinal cord, heart, lung, and kidney (FIG. 12). All othertissues appeared negative for α10 expression. These results point to arestricted distribution of the α10 integrin subunit.

Example 11

Immunohistochemical staining of α10 in fascia around tendon and skeletalmuscle and in tendon structures in heart valves.

Materials and Methods

Frozen sections of adult cartilage (trochlear groove) obtained duringsurgery (provided by Anders Lindahl, Salgrenska Hospital, Gothenburg,Sweden and frozen sections from of 3 day old mouse limb were fixed andprepared for immunohistochemistry as earlier described (Camper et al,JBC, 273, 20383–20389 (1998)). Expression of α10 integrin subunit wasanalysed using the polyclonal antibody against the cytoplasmic domain asa primary antibody (see Example 6) and a secondary antibody conjugatedto peroxidase.

Results

As shown in FIG. 13 expression of α10 was found in the fasciasurrounding tendon (a) and skeletal muscle (b) and in the tendonstructures in the heart valves (c). This localisation suggest that α10can bind to other matrix molecules in addition to the cartilage specificcollagen type II. The localisation of the integrin α10 on the surface oftendons indicate that α10 can be involved in unwanted adhesion thatoften occurs between tendon/ligaments and the surrounding tissue afterinfection, injury or after surgery.

Example 12

mRNA expression of. α10 integrin subunit in chondrocytes, endothelialcells and fibroblasts.

Material and Methods

Isolation of mRNA, synthesis of cDNA and PCR amplification was done asearlier described (Camper et al, JBC, 273, 20383–20389 (1998)).

Results

FIG. 14 shows PCR amplification of α10 cDNA from human articularchondrocytes (lanes A6 and B1), human umbilical vein endothelial cells(lane A2), human fibroblasts (lane A4) and rat tendon (FIG. 14 b, laneB2). Lanes 1, 3, and 5 in FIG. 14 A show amplified fragmentscorresponding to the integrin subunit α2 in endothelial cells,fibroblasts and chondrocytes, respectively. cDNA-primers correspondingto the α10 sequence positions nt 2919–2943 (forward) and nt 3554–3578(reverse) (see FIG. 6) were used to amplify α10 cDNA from the differentcells. The figure shows that α10 was amplified in all three cell types.Two fragments of α10 was amplified which represent the intact form ofα10 (larger fragment) and a splice variant (smaller fragment). Thelarger fragment was dominating in chondrocytes while the smallerfragment was more pronounced in tendon (B2).

Example 13

Construction of α10 mammalian expression vector.

The full length protein coding sequence of α10 (combined from 3 clones,see FIG. 6) was inserted into the mammalian expression vector,pcDNA3.1/Zeo (Invitrogen). The vector contains SV40 promoter and Zeosinselection sequence. The α10 containing expression vector was transfectedinto cells that express the β1-integrin subunit but lack expression ofthe α10 subunit. Expression of the α10 integrin subunit on the cellsurface can be analysed by immunoprecipitation and/or flow cytometryusing antibodies specific for α10. The ligand binding capacity and thefunction of the inserted α10 integrin subunit can be demonstrated incell adhesion experiment and in signalling experiments.

Example 14

Construction of mammalian expression vector containing a splice variantof α10.

The full length protein coding sequence of the splice variant of α10 (nt2942–nt3055 deleted) was inserted into the mammalian expression vectorpcDNA3 (see Example 13). Expression and function of the splice variantcan be analysed as described in example 13 and compared with the intactα10 integrin subunit.

Example 15

Partial isolation and characterisation of the α10 integrin genomic DNA.

Material and Methods

Human α10 cDNA, isolated from the race1-containing plasmid using therestriction enzymes BamHI (GIBCO BRL) and NcoI (Boehringer Mannheim),was ³²P-labelled and used as a probe for screening of a mouse 129 cosmidlibrary (provided by Reinhard Fässler, Lund University). Positive cloneswere isolated and subcloned. Selected plasmids were purified andsequenced as described earlier (Camper et al, JBC, 273, 20383–20389(1998)) using T3, T7 and internal specific primers. Primerscorresponding to mouse genomic DNA were then constructed and used in PCRto amplify and identify the genomic sequence of α10 from the cosmidclones.

Results

FIG. 15 shows 7958 nt of the α10 gene. This partial genomic DNA sequenceof α10 integrin contains 8 exons, and a Kozak sequence. The mousegenomic α10 sequence was used to generate a targeting vector forknockout experiments.

Example 16

Upregulation of α10 integrin subunit in chondrocytes cultured inalginate beads.

Material and Methods

Human chondrocytes cultured in monolayer for 2 weeks were detached withtrypsin-EDTA and introduced into alginate beads. Chondrocytes culturedin alginate are known to preserve their phenotype while chondrocytescultured in monolayer are dedifferentiated. After 11 days chondrocytescultured either in alginate or on monolayer were isolated and surfacelabelled with ¹²⁵I. The α10 integrin subunit was then immunoprecipitatedwith polyclonal antibodies recognising the cytoplasmic domain of α10(see Example 6 and Camper et al, JBC, 273, 20383–20389 (1998)).

Results

As shown in FIG. 16 chondrocytes cultured in alginate beads (lanes 3 and4) upregulated their protein expression of α10β1. This was in contrastto chondrocytes cultured in monolayer (lanes 1 and 2) which had a verylow expression of α10β1. Immunoprecipitation with ab control antibody isshown in lanes 1 and 3. It is known that chondrocytes preserve theircartilage specific matrixproduction in alginate cultures but not inmonolayer culture which point to that alginate preserve the phenotype ofchondrocytes. These results support that α10 integrin subunit can beused as a marker for differentiated chondrocytes.

Example 17

Immunoprecipitation of the α10 integrin subunit from human smooth musclecells.

Material and Methods

Human smooth muscle cells were isolated from human aorta. After one weekin culture the cells were ¹²⁵I-labelled, lysed and immunoprecipitatedwith antibodies against the integrin subunit β1 (lane 1), α1(lane 2), α2(lane 3), α10 (lane 4), α3 (lane 5), control (lane 6) (FIG. 17). Theexperiment was done as described in Example 7.

Results

The α10 antibody precipitated two bands from the smooth muscle cellscorresponding to the α10 and the β1 integrin subunit (FIG. 17).

Example 18

Construction of bacterial expression vector containing sequence for α10splice region.

TABLE I (SEQ ID NOS: 18–21, respectively, in order of appearance)α10pfor 5′GTTCAGAACCTGGGTTGCTACGTTGTTTCCGGTCTGATC (SEQ ID No.: 18)ATCTCCGCTCTGCTGCCGGCTGT-3′ α10pfor25′GGGGCATATGGTTCAGAACCTGGGTTGCTACGTTG-3′ (SEQ ID No.: 19) α10prev5′GATAACCTGGGACAAGCTTAGGAAGTAGTTACCACCGT (SEQ ID No.: 20) GAGCAACAGCCGGCAGCAGAGCGGA-3′ α10prev2 5′GGGGGGATCCGCGCGGCACCAGGCCGCTGATAACCTGG(SEQ ID No.: 21) GACAAGCTTAGGAAGT-3′

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1. A recombinant or isolated collagen binding integrin subunit α10consisting of SEQ ID NO: 2 or a fragment thereof, wherein the fragmentis selected from the group consisting of amino acid 952 to amino acid986 of SEQ ID NO: 2, amino acid 140 to amino acid 337 of SEQ ID NO:2,and SEQ ID NO:
 7. 2. A recombinant or isolated collagen binding integrinsubunit α10 consisting of SEQ ID NO: 2 or a fragment thereof, whereinthe fragment is a peptide comprising SEQ ID NO:
 7. 3. A recombinant orisolated collagen binding integrin subunit α10 consisting of SEQ ID NO:2 or a fragment thereof, wherein the fragment is a peptide comprisingamino acid 952 to amino acid 986 of SEQ ID NO:
 2. 4. A recombinant orisolated collagen binding integrin subunit α10 consisting of SEQ ID NO:2 or a fragment thereof, wherein the fragment is a peptide comprisingamino acid 140 to amino acid 337 of SEQ ID NO:
 2. 5. A recombinant orisolated collagen binding integrin subunit α10 comprising SEQ ID NO: 2or a fragment thereof, wherein the fragment is amino acid 952 to aminoacid 986 of SEQ ID NO: 2, amino acid 140 to amino acid 337 of SEQ ID NO:2, or SEQ ID NO:
 7. 6. A recombinant or isolated collagen bindingintegrin subunit α10 consisting of SEQ ID NO: 2 or a fragment thereof,wherein the fragment is a peptide consisting of SEQ ID NO:7.
 7. Arecombinant or isolated collagen binding integrin subunit α10 consistingof SEQ ID NO: 2 or a fragment thereof, wherein the fragment is a peptideconsisting of amino acid 952 to amino acid 986 of SEQ ID NO:2.
 8. Arecombinant or isolated collagen binding integrin subunit α10 consistingof SEQ ID NO: 2 or a fragment thereof, wherein the fragment is a peptideconsisting of amino acid 140 to amino acid 337 of SEQ ID NO:2.
 9. Arecombinant or isolated collagen binding integrin subunit α10 consistingof SEQ ID NO:4 or a fragment thereof wherein the fragment is selectedfrom the group consisting of amino acid 140 to amino acid 337 of SEQ IDNO:2 and SEQ ID NO:7.
 10. A recombinant or isolated collagen bindingintegrin subunit α10 comprising SEQ ID NO: 4 or a fragment thereofwherein the fragment is selected from the group consisting of amino acid140 to amino acid 337 of SEQ ID NO:2 and SEQ ID NO:7.